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PubMed2003

The low viral production in trophoblastic cells is due to a high endocytic internalization of the human immunodeficiency virus type 1 and can be overcome by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-1.

Maternal-infant transmission of human immunodeficiency virus type-1 (HIV-1) is the primary cause of this retrovirus infection in neonates. Trophoblasts have been proposed to play a critical role in modulating virus spread to the fetus. This paper addresses the mechanism of HIV-1 biology in trophoblastic cells. The trophoblastic cell lines BeWo, JAR, and JEG-3 were infected with reporter HIV-1 particles pseudotyped with envelope glycoproteins from the vesicular stomatitis virus or various strains of HIV-1. We demonstrate that despite a high internalization process of HIV-1 and no block in viral production, HIV-1 established a limited infection of trophoblasts with the production of very few progeny viruses. The factor responsible for this restriction to virus replication in such a cellular microenvironment is that the intracellular p24 is concentrated predominantly in endosomal vesicles following HIV-1 entry. HIV-1 transcription and virus production of infectious particles were both augmented upon treatment of trophoblasts with tumor necrosis factor-alpha and interleukin-1. However, the amount of progeny virions released by trophoblasts infected with native HIV-1 virions was so low even in the presence of pro-inflammatory cytokines that a co-culture step with indicator cells was necessary to detect virus production. Collectively these data illustrate for the first time that the natural low permissiveness of trophoblasts to productive HIV-1 infection is because of a restriction in the mode of entry, and such a limitation can be overcome with physiologic doses of tumor necrosis factor-alpha and interleukin-1, which are both expressed by the placenta, in conjunction with cell-cell contact. Considering that there is a linear correlation between viral load and HIV-1 vertical transmission, the environment may thus contribute to the propagation of HIV-1 across the placenta.

Cell Line↗

PubMed1980

Oligonucleotide sequence analyses indicate that vesicular stomatitis virus large defective interfering virus particle RNA is made by internal deletion: evidence for similar transcription polyadenylation signals for the synthesis of all vesicular stomatitis virus mRNA species.

RNase T(1) oligonucleotide fingerprint analyses of three vesicular stomatitis virus Indiana serotype small defective interfering (DI) particle RNA species indicate that they only have oligonucleotides derived from the 5' region of the viral genome. These studies also indicate that these three DI RNAs have partial L gene sequences as well as two 5' viral oligonucleotides (59 and 70) that are not transcribed into L (or other) mRNA species (J. P. Clewley and D. H. L. Bishop, J. Virol. 30:116-123, 1979). Analyses of the large DI RNA (LT DI) reveal a different origin. The LT DI RNA has oligonucleotides derived from both the 3' end of the genome (including all the large oligonucleotides identified for N, NS, M, and G genes), in addition to at least one of the 5'-proximal L gene oligonucleotides (47), as well as all seven oligonucleotides (3, 38, 42, 43, 44B, 59, and 70) that are not protected from nuclease digestion after the formation of mRNA-viral RNA duplexes (Clewley and Bishop). It appears therefore that the genesis of LT RNA involves a deletion of internal L gene sequences from the viral RNA. Oligonucleotide sequence analyses have been undertaken on several of the vesicular stomatitis viral RNA oligonucleotides, including all seven (3, 38, 42, 43, 44B, 59, and 70) that are not transcribed into mRNA. The analyses confirm that oligonucleotides 59 [3'...GAACACCAAAAAUAAAAAAUA(G)...5'] and 70 [3'...GACCAAAACACCA(G)...5'] are at the 5'-end region of the viral genome. Oligonucleotide 38 [3'...GAAAUUCAUACUUUUUU(U)(G)...5'] may represent the termination signal for L mRNA synthesis (R. A. Lazzarini, personal communication). Oligonucleotide 43 [3'...GUAUACUUUUUUU(G)...5'] corresponds to the sequence shown to be the N gene mRNA polyadenylation signal (D. J. McGeoch, Cell 17:673-681, 1979). The other three oligonucleotides share a common feature with oligonucleotides 43 and 38, viz., a stretch of 6 or 7 U residues preceded by an AUAC sequence. Thus the sequence of oligonucleotide 3 is 3'...GAAUUAAUAUAAAAUUAAAAAUUAAAAAUACUUUUUU(U)(G)...5', whereas that of oligonucleotide 42 is 3'...GAUACUUUUUUUCAU(U)(G)...5', and that of oligonucleotide 44B is 3'...G(U)AUACUUUUUU(G)...5'. These sequence analyses suggest a common polyadenylation signal for the synthesis of all vesicular stomatitis virus mRNA species, i.e., the sequence (3')...AUACUUUUUU(U)...(5').

Animals↗

PubMed2000

Surveillance of influenza viruses isolated from travellers at Nagoya International Airport.

In order to conduct a survey of influenza viruses entering Japan via travellers arriving by airplanes, gargle solutions were collected from passengers who reported to the quarantine station of Nagoya International Airport complaining of respiratory symptoms. From 504 samples collected between August 1996 and March 1999, 30 influenza virus strains were isolated. Twenty-eight of the isolates were influenza A (H3N2) viruses and two were influenza B viruses. No H1N1 virus was isolated. Among 28 isolates of H3N2 virus, 3 strains were obtained outside the influenza season. Nucleotide sequences of the haemagglutinin (HA) genes of these isolates along with those from domestic patients were analysed in order to determine the influence of imported influenza viruses by travellers on epidemics in Japan. From the phylogenetic and chronological aspects, the possibility was suggested in one case in 1997/8 and two in the 1998/9 season that imported virus by travellers may have influenced the domestic influenza epidemics.

Adolescent↗

PubMed1993

Molecular identification of cardioviruses by enzymatic amplification.

Cardiovirus specific sequences located in the 5' NTR are used to amplify viral RNA by PCR. General primers were selected for the amplification of cardioviruses, including encephalomyocarditis virus (EMCV), mengovirus and Theiler's murine encephalomyelitis virus (TMEV. Additionally, ECHO virus type 22, an atypical enterovirus, could also be detected with the general cardiovirus primers. An internal encephalomyocarditis virus specific probe and a general cardiovirus probe are used to discriminate EMCV from other cardioviruses. The sensitivity of the PCR was determined at 10 pfu after hybridization with an internal oligonucleotide probe. Specificity was tested with a broad range of picornaviruses and other nonrelated RNA and DNA viruses. The applicability of the assay was demonstrated by the detection of TMEV RNA in tissue samples from experimentally-infected mice.

Animals↗

PubMed1990

Structural requirements for C3d,g/Epstein-Barr virus receptor (CR2/CD21) ligand binding, internalization, and viral infection.

The structure of CR2, the human C3d,g/EBV receptor (CR2/CD21) consists of 15 or 16 60-70 amino acid repeats called short consensus repeats (SCRs) followed by a transmembrane and a 34-amino acid intracytoplasmic domain. Functions of CR2 include binding the human complement component C3d,g when it is covalently attached to targets or cross-linked in the fluid phase. In addition, CR2 binds the Epstein-Barr virus (EBV) and mediates internalization of EBV and subsequent infection of cells. In order to explore functional roles of the repetitive extracytoplasmic SCR structure and the intracytoplasmic domain of CR2, we have created truncated CR2 (rCR2) mutants bearing serial deletions of extracytoplasmic SCRs and also the intracytoplasmic tail. We then stably transfected these rCR2 mutants into two cell lines, murine fibroblast L cells and human erythroleukemic K562 cells. Phenotypic analysis of these expressed mutants revealed that 1) The C3d,g- and EBV-binding sites are found in the two amino-terminal SCRs of CR2, 2) expression of SCRs 3 and 4 is further required for high affinity binding to soluble cross-linked C3d,g, 3) the intracytoplasmic domain of CR2 is not required for binding C3d,g or EBV but is necessary for internalization of cross-linked C3d,g as well as for EBV infection of cells, 4) monoclonal anti-CR2 antibodies with similar activities react with single widely separated epitopes, and 5) no functional roles can yet be clearly assigned to SCRs 5-15, as rCR2 mutants not containing these SCRs show no major differences from wild-type rCR2 in binding or internalizing cross-linked C3d,g or mediating EBV binding and infection.

Animals↗

PubMed1995

Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.

The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.

Animals↗

PubMed2025

The Single Amino Acid Change of R516K Enables Efficient Generation of Vesicular Stomatitis Virus-Based Crimean-Congo Hemorrhagic Fever Reporter Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a medically important tick-borne virus, causing severe hemorrhagic diseases in humans. There are no approved vaccines and therapeutics for CCHFV infection. The study of CCHFV authentic virus requires biosafety level 3 facilities, hindering the research and development of antivirals. Here we report the generation of a recombinant vesicular stomatitis virus (VSV) bearing both CCHFV glycoprotein precursor (GPC) and EGFP reporter (rVSV-CCHFV-GFP). We also find that the acquisition of an unexpected single R516K mutation in the GPC protein enables the packaging of high-titer pseudotyped particles. The replication-competent rVSV-CCHFV-GFP reporter virus resembles the entry properties of the authentic virus and allows for rapid assessment of susceptible cell lines, neutralizing antibodies, and host entry factors such as heparan sulfate in fluorescence-based assays. This study provides a valuable strategy for packaging of high-titer CCHFV pseudovirus, and the tool generated here can be served for the identification and evaluation of countermeasures against the cell entry of CCHFV.

Hemorrhagic Fever Virus, Crimean-Congo↗

PubMed1990

Mechanisms of transmissible gastroenteritis coronavirus neutralization.

Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies of a single specificity [monoclonal antibodies (MAbs)]. Most of the virus was neutralized in the first 2-3 min of a reversible reaction, which was followed by a second phase with a decreased neutralization rate and, in some cases, by a persistent fraction, which was a function of the MAb and of the antibody-to-virus ratio. Neutralization of TGEV is a specific event that requires the location of the epitope involved in the neutralization in the appropriate structural context, which is present in the wild-type virus but not in certain MAb escaping mutants. In neutralization of TGEV by binary combinations of MAbs specific for the same or for different antigenic sites, either no cooperation or a synergistic effect, respectively, was observed. Mechanisms of TGEV neutralization by MAbs were characterized at high, intermediate, and low antibody-to-virus ratios. Under these conditions, mainly three steps of the replication cycle were inhibited: binding of virus to the cell, internalization, and a step that takes place after internalization. In addition, virus aggregation could be responsible for the neutralization of 10 to 20% of virus infectivity.

Animals↗

PubMed1985

Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro.

Pathogenic parental rabies virus and apathogenic variant virus were shown to differ in their ability to infect neurons in vivo and neuroblastoma cells in vitro. After intracerebral inoculation, the distribution of infected neurons in the brain was similar for both viruses, but the rate of spread throughout the brain, the number of infected neurons, and the degree of cellular necrosis were much lower in the case of apathogenic virus. After adsorption to mouse neuroblastoma cells, apathogenic virus was less rapidly internalized than pathogenic virus, and cell-to-cell spread of apathogenic variant virus was completely prevented by the addition of rabies virus-neutralizing antibody, whereas the spread of pathogenic virus was not affected.

Animals↗

PubMed2004

Human rhinovirus type 2-antibody complexes enter and infect cells via Fc-gamma receptor IIB1.

HeLa cells were stably transfected with a cDNA clone encoding the B1 isoform of the mouse FcgammaRII receptor (hereafter referred to as HeLa-FcRII cells). The receptor was expressed at high level at the plasma membrane in about 90% of the cells. These cells bound and internalized mouse monoclonal virus-neutralizing antibodies 8F5 and 3B10 of the subtype immunoglobulin G2a (IgG2a) and IgG1, respectively. Binding of the minor-group human rhinovirus type 2 (HRV2) to its natural receptors, members of the low-density lipoprotein receptor family, is dependent on the presence of Ca(2+) ions. Thus, chelating Ca(2+) ions with EDTA prevented HRV2 binding, entry, and infection. However, upon complex formation of (35)S-labeled HRV2 with 8F5 or 3B10, virus was bound, internalized, and degraded in HeLa-FcRII cells. Furthermore, challenge of these cells with HRV2-8F5 or HRV2-3B10 complexes resulted in de novo synthesis of viral proteins, as shown by indirect immunofluorescence microscopy. These data demonstrate that minor-group receptors can be replaced by surrogate receptors to mediate HRV2 cell entry, delivery into endosomal compartments, and productive uncoating. Consequently, the conformational change and uncoating of HRV2 appears to be solely triggered by the low-pH (pH </= 5.6) environment in these compartments.

Animals↗

PubMed1993

Interaction of hydrophobized antiviral antibodies with influenza virus infected MDCK cells.

The ability of artificially stearoylated antibodies to influenza virus hemagglutinin and M1 proteins to interfere with influenza infection in MDCK cells has been studied. Both the modified anti-hemagglutinin (polyclonal) and anti-M1 (monoclonal) antibodies neutralize the virus when added before the infection. The effect can be attributed to the interaction of stearoylated antibodies with the virus surface, which is enhanced by fatty acylation (anti-hemagglutinin), or to the antibody uptake in the cell endocytic compartments simultaneously with the virion which permits antibodies to interact with the virus envelope internal antigen (anti-M1). The stearoylated antibodies to hemagglutinin inhibit the virus reproduction being added to the infected cells. This effect is believed to be due to the interaction of the antibodies with newly synthesized hemagglutinin on the cell surface which disturbs the virus budding and assemblage; fatty acylation intensifying this interaction.

Acylation↗

PubMedDate not supplied

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA.

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.

DNA, Recombinant↗

PubMed1998

Hepatitis C in France: a national survey in the Departments of Internal Medicine and Infectious Diseases. The GERMIVIC (Joint Study Group on Hepatitis C virus of the French National Society of Internal Medicine and the French Society of Infectious Diseases).

Infection with Hepatitis C virus is a significant public health problem that has important clinical and financial consequences. Understanding of the epidemiology of HCV is needed to help define future therapeutic and preventive strategies. So far, the importance and characteristics of the epidemics have been best appreciated in specialist units dealing with liver disease. The purpose of our study was to survey the number and characteristics of hepatitis C antibody positive patients in Departments of Internal Medicine and Infectious Diseases. We conducted a multicentre national prospective analysis of all positive HCV-antibody patients, either inpatient or outpatient, reported over a period of one month across France. Two thousand and two cases were identified. Epidemiological, clinical and therapeutic characteristics are described. Risk factors were identified in 86%. For 10% of the patients, hepatitis C seropositivity was discovered during the period of survey. At the time of first diagnosis, 47% of patients presented with no clinical or biological abnormality. Coinfection with HIV was frequent (59%). Only 20.3% of the patients had received or were receiving a treatment with interferon. Within the limits of the methodology used, this study shows that Hepatitis C infection is a substantial clinical problem in French Departments of Internal Medicine and Infectious Diseases. Our findings may help the public health authorities in better appreciating the impact of hepatitis C and making policy decisions.

Adolescent↗

PubMed1995

Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing. The International HCV Collaborative Study Group.

Variation in the 5' non-coding region (5'NCR) of hepatitis C virus (HCV) was investigated in detail by comparing 314 5'NCR sequences of viruses of genotypes 1 to 6. Evidence was obtained for the existence of associations between particular 5'NCR sequence motifs and virus types and subtypes. No recombination was observed between the 5'NCR and coding regions of different genotypes, implying that the sequence of subgenomic regions such as the 5'NCR can be used to deduce virus genotype. The distribution of polymorphic sites within the 5'NCR is used to propose improved oligonucleotide primers for virus detection and quantification that would be equally efficient in detecting RNA of different virus genotypes. The accuracy of two different genotyping methods (RFLP and the line probe assay) based on analysis of sequence polymorphisms in the 5'NCR is predicted from the sequences surveyed to be 97% and 83% respectively for types 1 to 6, with higher accuracies for distinguishing between subtypes 1a/1b, 2a/2b or 3a/3b. Several sites of genotype-specific polymorphism were covariant and maintained the base pairings required for a secondary structure model of the 5'NCR. Other sites of variation suggest minor modifications to this model and have implications for the probable functions of the 5'NCR.

Base Composition↗

PubMed1990

cis rescue of a mutated reverse transcriptase gene of human hepatitis B virus by creation of an internal ATG.

Using mutational analysis, we have investigated the translation strategy of the reverse transcriptase gene (pol) of human hepatitis B virus. It has been proposed that this pol gene product is synthesized as a core-pol fusion protein from a polycistronic mRNA template via ribosomal frameshifting, a mechanism often seen in retroelements. Our data indicate that creation of a novel ATG initiation codon near the original ATG can compensate for a lethal missense mutation at the first ATG position of the pol open reading frame. Genetic analysis has rigorously ruled out the possibilities of frameshifting, non-ATG initiation, or RNA editing. These results are discussed in the context of a 5'-end entry model versus a novel model of direct internal entry of ribosomes.

Base Sequence↗

PubMedDate not supplied

Assessment of retrovirus-expressed nucleoprotein as a vaccine against lethal influenza virus infections of chickens.

Hemagglutinin-based influenza vaccines stimulate protection in chickens that is limited to the serotype of the expressed hemagglutinin. To evaluate whether a more highly conserved influenza virus protein might stimulate a broader protective response, the influenza virus nucleoprotein (NP) was introduced into a retroviral vector (mRCAS/NP). NP is an internal influenza virus protein that has been shown to stimulate cytotoxic T-cell responses in influenza-virus-infected mice. Cells infected with mRCAS/NP expressed approximately 10% of the level of NP observed in influenza-virus-infected chicken embryo fibroblasts. Immunocompetent chicks were vaccinated intramuscularly with approximately 1 x 10(5) NP-expressing units of mRCAS/NP. Four weeks later, chicks were bled and challenged with a highly pathogenic avian influenza virus (A/Chicken/Victoria/1/85). The NP-expressing vector stimulated an influenza-virus-specific response, as indicated by the presence of antibody to NP, but failed to protect against the lethal challenge.

Animals↗

PubMed1996

Degradation of mutant influenza virus hemagglutinins is influenced by cytoplasmic sequences independent of internalization signals.

A mutant influenza virus hemagglutinin, HA+8, having a carboxyl-terminal extension of 8 amino acids that included 4 aromatic residues, was internalized within 2 min of arriving at the cell surface and was degraded quickly by a process that was inhibited by ammonium chloride. Through second-site mutagenesis, the internalization sequence of HA+8 was found to closely resemble the internalization signals of the transferrin receptor or large mannose 6-phosphate receptor. Comparison of the intracellular traffic of HA+8 and a series of other HA mutants that differed in their rates of internalization revealed a relation between the amount of the protein on the plasma membrane at steady state and the internalization rate that would be predicted if most of each protein recycled to the cell surface. However, there was no simple correlation between the internalization rate and the rate of degradation, indicating that transport to the compartment where degradation occurred was not simply a function of the concentration of the proteins in early endosomes. The internal populations of both HA+8, which was degraded with a t1/2 of 1.9 h, and HA-Y543, which was degraded with a t1/2 of 2.9 h, were found by cell fractionation and density-shift experiments to reside in early endosomes with little accumulation in lysosomes. A fluid-phase marker reached lysosomes 3-4-fold faster than these proteins were degraded. Degradation of these mutant HAs involved a rate-determining step in early endosomes that was sensitive to some feature of the protein that depended upon sequence differences in the cytoplasmic domain unrelated to the internalization signal.

Amino Acid Sequence↗

PubMed1999

Internal processing of hepatitis C virus NS3 protein.

Hepatitis C virus (HCV) NS3 protein contains at least three enzymatic activities: NS2-3 protease, NS3 serine protease, and NTPase/RNA helicase. It has been shown that NS2/3 cleavage is mediated by NS2-3 protease, whereas NS3 serine protease is responsible for the other four cleavage sites of the nonstructural (NS) region. In this study, we showed that the internal cleavage of NS3 protein produced two products of 49 kDa (NS3a) and 23 kDa (NS3b) when the entire NS3 region (aa 1027-1657) or the whole open reading frame (aa 1-3010) was expressed in mammalian and insect cells. By means of site-directed mutagenesis, we demonstrated that NS3a/NS3b cleavage occurs within the RNA helicase sequence motif that is highly conserved in the Flaviviridae family and that neither NS2-3 protease nor NS3 serine protease was responsible for this cleavage. The NS3 protease of flaviviruses, dengue virus type 2, for example, has been shown to mediate the internal cleavage of NS3. The NS3 proteins of HCV and dengue virus may thus be cleaved internally at the same sequence by different mechanisms of proteolysis. Also discussed is a possible role for the internal processing of HCV NS3 in the viral life cycle and its pathogenesis.

Adenosine Triphosphatases↗